1. Field of the Invention
This invention is in the field of cancer therapy and in particular, relates to compositions and methods to specifically kill tumor cells by the production of toxic compounds in the tumor cells.
2. Description of the Related Art
Inefficiency of gene delivery, together with inadequate bystander killing, represent two major conceptual hurdles in the development of a toxin mediated gene therapy for human malignancy. Gene transfer is a useful adjunct in the development of new therapies for human malignancy. Tumor cell expression of histocompatibility antigens, cytokines, or growth factors (for example, IL-2, IL-4, GMCSF) appears to enhance immune-mediated clearance of malignant cells in animal models, and expression of chemo-protectant gene products, such as p-glycoprotein in autologous bone marrow cells, is under study as a means of minimizing marrow toxicity following administration of otherwise lethal doses of chemotherapeutic agents.
Theoretically, the most direct mechanism for tumor cell killing using gene transfer is the selective expression of cytotoxic gene products within tumor cells. However, no recombinant enzyme or toxin has proven useful in mediating high levels of toxicity in unselected tumor cells. Classical enzymatic toxins such as pseudomonas exotoxin A, diphtheria toxin and ricin are unlikely to be useful in this context, since these enzymes kill only cells in which they are expressed, and no currently available gene transfer vector is capable of gene delivery to a sufficiently high percentage of tumor cells to make use of the above recombinant enzymes.
Another strategy that has been developed to selectively kill tumor cells involves the delivery and expression of the HSV dThd kinase gene to replicating tumor cells followed by treatment with ganciclovir. Ganciclovir is readily phosphorylated by the HSV dThd kinase, and its phosphorylated metabolites are toxic to the cell. Very little phosphorylation of ganciclovir occurs in normal human cells. Although only those cells expressing the HSV dThd kinase should be sensitive to ganciclovir (since its phosphorylated metabolites do not readily cross cell membranes), in vitro and in vivo experiments have shown that a greater number of tumor cells are killed by ganciclovir treatment than would be expected based on the percentage of cells containing the HSV dThd kinase gene. This unexpected result has been termed the “bystander effect” or “metabolic cooperation.” It is thought that the phosphorylated metabolites of ganciclovir may be passed from one cell to another through gap junctions. However, even if a nucleoside monophosphate such as ganciclovir monophosphate were released into the medium by cell lysis, the metabolite would not be able to enter neighboring cells and would likely be degraded (inactivated) to the nucleoside by phosphatases.
Although the bystander effect has been observed in initial experiments using HSV dThd kinase, the limitations of current gene delivery vehicles mean that a much greater bystander effect is important to successfully treat human tumors using this approach. One difficulty with the current bystander toxicity models is that bystander toxicity with metabolites that do not readily cross the cell membrane will not be sufficient to overcome a low efficiency of gene transfer (for example, transfection, transduction, etc.).
One protocol for treating brain tumors in humans uses retroviral delivery of HSV dThd kinase, followed by ganciclovir administration. In rat models, using HSV dThd in this context, tumor regressions have been observed. The HSV dThd kinase approach has not proven sufficient in humans thus far; this may in part be due to (1) inadequate bystander toxicity with HSV dThd kinase, and (2) cell killing only of dividing cells using HSV dThd kinase with ganciclovir. The usefulness of E. coli cytosine deaminase, which converts 5-fluorocytosine to 5-fluorouracil, has recently been reported to provide substantial bystander toxicity. However, 5-FU is not a highly toxic compound in this setting and bystander killing in vitro has been inefficient, i.e., similar to that of observed with HSV dThd kinase.
Prodrug activation by an otherwise non-toxic enzyme (for example, HSV dThd kinase, cytosine deaminase) has advantages over the expression of directly toxic genes, such as ricin, diphtheria toxin, or pseudomonas exotoxin. These advantages include the capability to (1) titrate cell killing, (2) optimize therapeutic index by adjusting either levels of prodrug or of recombinant enzyme expression, and (3) interrupt toxicity by omitting administration of the prodrug. However, like other recombinant toxic genes, gene transfer of HSV dThd kinase followed by treatment with ganciclovir is neither designed to kill bystander cells nor likely to have broad bystander toxicity in vivo.
An additional problem with the use of the HSV dThd kinase or cytosine deaminase to create toxic metabolites in tumor cells is the fact that the agents activated by HSV dThd kinase (ganciclovir, etc.) and cytosine deaminase (5-fluorocytosine) kill only cells synthesizing DNA. Even if a considerable number of nontransfected cells are killed, one would not expect to kill the nondividing tumor cells with these agents.
Thus, there exists a need for a toxin gene therapy method that overcomes the problems of inefficient gene delivery, cell replication-dependent killing and low toxin diffusion between cells. The present invention fulfills this longstanding need and desire in the art.